title: Anti-donor HLA class I antibodies: pathways to endothelial cell activation and cell-mediated allograft rejection.
abstract: BACKGROUND
  The development of donor-specific human leukocyte antigen (HLA) class I antibodies after organ transplantation is associated with subsequent acute and chronic rejection. The aim of this study was to examine the role of anti-HLA class I antibody in modulating endothelium-leukocyte interaction.
  
  
  METHODS
  Human microvascular endothelial cells (HMEC-1) stimulated with HLA class I antibody (W6/32) or allospecific antibodies from sensitized patients (n=6) were examined for activation of transcription factor CREB by Western blotting. Up-regulation of endothelial adhesion molecules and chemokines was measured by flow cytometry and quantitative polymerase chain reaction, respectively. Leukocyte adhesion was evaluated by chemotaxis and in vitro flow-based assays.
  
  
  RESULTS
  Treatment of HMEC-1 cells with HLA class I antibody resulted in the phosphorylation of CREB in protein kinase A-dependent pathway. Furthermore, there was a significant increase in the expression of cell surface VCAM-1 (Akt-dependent) and ICAM-1 in Akt-dependent and extracellular signal-regulated kinase-dependent manner (P<0.001). Additionally, exposure to W6/32 antibody induced significant expression of interleukin-6, CXCL8, CXCL10, and CCL5. Knockdown of CREB produced a reduction in W6/32-induced CXCL8 expression (P<0.001). Media from W6/32-treated endothelial cells induced a significant monocyte chemotaxis (P<0.001) and flow-based adhesion assay demonstrated an increase in monocyte adhesion to endothelial cells compared with the control group (P<0.001). Importantly, allospecific antibodies from sensitized patients also activated endothelial CREB and significantly up-regulated VCAM-1, ICAM-1, and CXCL8.
  
  
  CONCLUSIONS
  These findings suggest that donor-specific HLA class I antibodies directly activate endothelial cells leading to an increase in their potential to recruit and bind recipient leukocytes, thereby increasing the potential for allograft inflammation.
authors.affiliation: Applied Immunobiology and Transplantation Group, Institute of Cellular Medicine, Medical School, University of Newcastle Upon Tyne, Newcastle Upon Tyne, UK.
authors.forename: Fatmah M A
authors.initials: FM
authors.lastname: Naemi
authors.forename: Vaughan
authors.initials: V
authors.lastname: Carter
authors.forename: John A
authors.initials: JA
authors.lastname: Kirby
authors.forename: Simi
authors.initials: S
authors.lastname: Ali
mesh_terms: D002460:Cell Line; D002634:Chemotaxis, Leukocyte; D017362:Cyclic AMP Response Element-Binding Protein / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000502:physiology; D016207:Cytokines / Q000235:genetics; D042783:Endothelial Cells / Q000502:physiology*; D048049:Extracellular Signal-Regulated MAP Kinases / Q000502:physiology; D006084:Graft Rejection / Q000209:etiology*; D015395:Histocompatibility Antigens Class I / Q000276:immunology*; D006801:Humans; D007518:Isoantibodies / Q000502:physiology*; D019869:Phosphatidylinositol 3-Kinases / Q000502:physiology; D010766:Phosphorylation; D014184:Transplantation, Homologous
chemical_list: C115163:CREB1 protein, human; D017362:Cyclic AMP Response Element-Binding Protein; D016207:Cytokines; D015395:Histocompatibility Antigens Class I; D007518:Isoantibodies; D019869:Phosphatidylinositol 3-Kinases; D017868:Cyclic AMP-Dependent Protein Kinases; D048049:Extracellular Signal-Regulated MAP Kinases